Oxyntomodulin peptide analogue

ABSTRACT

The present invention provides Oxyntomodulin peptide analogues useful in the treatment of diabetes and/or obesity.

The present invention relates to Oxyntomodulin peptide analogues and toPEGylated derivatives thereof for use in treating diabetes and/orobesity.

Oxyntomodulin (OXM) is a 37 amino acid peptide hormone that is releasedalong with Glucagon-Like-Peptide 1 (GLP-1) from the L-cells of the smallintestine in proportion to nutrient ingestion. It is composed of thefull 29 residue sequence of glucagon (Gcg) with an octapeptide extensionat the C-terminus as a result of tissue-specific alternate processing ofpreproglucagon. Endogenous OXM is quickly degraded in vivo by dipeptidylpeptidase IV and other peptidases.

Distinct receptors for OXM have not yet been identified. OXM binds toand fully activates both the GLP-1 receptor (GLP-1R) and the glucagonreceptor (GcgR) in vitro with similar potencies at the two receptors.

OXM is involved in regulation of food intake and body weight. Acuteadministration of OXM to normal-weight human subjects reduced hunger anddecreased meal size by 19%. In a 4-week study with overweight and obesesubjects, three times daily preprandial subcutaneous administration ofOXM produced a weight loss of 2.3 kg compared with 0.5 kg in the placebogroup. In this trial, nausea, the most common side effect associatedwith GLP-1 based therapy (such as exenatide and liraglutide), wassignificantly less prevalent. OXM increased energy usage throughpromotion of increased physical activity in overweight and obese humans,although the mechanism of the effect is unclear.

OXM presents several challenges for development into acommercially-viable therapeutic agent. As mentioned above, it is rapidlydegraded in vivo as well as being subjected to rapid renal clearance dueto its small size. It is therefore desirable to identify OXM peptideanalogues with improved metabolic stability and reduced rate ofclearance. Furthermore, the GcgR agonist activity inherent in OXMpresents a risk of negatively impacting glycemic control. Thus, it isalso desirable to optimize the potency of an OXM peptide analoguedesigned for therapeutic use while maintaining an appropriate balancebetween activities at the GLP-1R and GcgR. Activation of GLP-1R isresponsible for an insulinotropic effect while activation of both GLP-1Rand GcgR may play a role in the weight loss effects. It is thereforedesirable to produce an OXM peptide analogue which has potentinsulinotropic activity and promotes weight loss such that it can beused for the treatment of non-insulin dependent diabetes and/or obesity.

OXM peptides with amino acid substitutions to improve stability and withadditional modifications to slow clearance, such as PEGylation orlipidation are disclosed in WO 2008101017, WO2006134340, WO2007100535,and Pocai et al. Diabetes 58:2258-2266, 2009. While these OXM-derivedpeptides may represent a potential improvement over the wild typepeptide, the doses required to achieve a sizable weight reduction in adiet-induced obese (DIO) mouse model are typically higher than may beconsidered feasible for pharmaceutical commercialization. For example,Pocai et al (2009) reported an average 11 g (˜25%) weight loss after 13days of dosing with 1900 nmol/kg (˜8 mg/kg) every other day (QOD).

Despite the availability of various OXM peptides and analogues thereof,there is still a need for more potent, stable, long-acting, andwell-tolerated OXM peptide analogues having a ratio of GcgR/GLP-1Ractivity which has been optimized such that the potency andinsulinotropic activity of the peptide provides effective treatments fordiabetes, preferably type 2 diabetes and related disorders. It is alsodesirable to provide OXM peptide analogues thereof which provideeffective treatments to reduce body weight. Accordingly, the presentinvention seeks to provide effective treatments for diabetes and/orobesity.

The present invention comprises an OXM peptide analogue with amino acidsubstitutions introduced to optimize metabolic stability and modulatethe relative GcgR/GLP-1R activities while optimizing overall potency. Inaddition, the OXM peptide analogue of the present invention is PEGylatedat selected positions for enhancement of time action thereby allowingfor less frequent dosing.

The present invention provides an Oxyntomodulin peptide analoguecomprising the amino acid sequence:

1                    5                      10His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-                 15                  20         Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Gln-Glu-Phe-         25                    30Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-      35Asn-Asn-Ile-Ala-Xaa₃₈-Xaa₃₉ (SEQ ID NO: 5)wherein Xaa₃₈ is Cys, Cys-PEG, or is absent, Xaa₃₉ is Cys, Cys-PEG, oris absent, and wherein the C-terminal amino acid is optionally amidated.

The present invention provides an Oxyntomodulin peptide analoguecomprising the amino acid sequence:

1                    5                      10His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-                 15                  20Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Gln-Glu-Phe-         25                    30Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-      35 Asn-Asn-Ile-Ala.(SEQ ID NO: 1)

Furthermore, the present invention provides an Oxyntomodulin peptideanalogue comprising the amino acid sequence:

1                    5                      10His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-                 15                  20Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Gln-Glu-Phe-         25                    30Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-      35Asn-Asn-Ile-Ala-Cys-Cys (SEQ ID NO: 2)wherein the Cys at position 38 is optionally PEGylated, and wherein theCys present at position 39 is optionally PEGylated and the carboxylgroup of the Cys at position 39 is optionally amidated.

Preferably, the Oxyntomodulin peptide analogue of SEQ ID NO: 2 isPEGylated on either the Cys at position 38 or the Cys at position 39 orboth with a 40 kDa PEG molecule covalently linked to the thiol group ofthe Cys residue at these positions. More preferably the Oxyntomodulinpeptide analogue is PEGylated on each Cys residue at both position 38and position 39 with a 20 kDa PEG molecule covalently linked to eachthiol group of each Cys residue at these positions. Optionally, the Cysresidue at position 39 may be absent from SEQ ID NO: 2, leaving a singlesite for PEGylation at position 38.

The more preferred Oxyntomodulin peptide analogue comprises the aminoacid sequence:

1                    5                      10His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-                 15                  20Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Gln-Glu-Phe-         25                    30Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-      35Asn-Asn-Ile-Ala-Cys(20 kDa PEG)- Cys(20 kDa PEG) (SEQ ID NO: 3)wherein the carboxyl group of the PEGylated Cys at position 39 isoptionally amidated.

The most preferred Oxyntomodulin peptide analogue comprises the aminoacid sequence of SEQ ID NO: 3, wherein the carboxyl group of thePEGylated Cys at position 39 is amidated.

The PEG molecule used in the present invention may be linear or branchedand is preferably a linear PEG molecule.

The present invention provides a pharmaceutical composition comprisingthe Oxyntomodulin peptide analogue as defined above, and apharmaceutically acceptable carrier, diluent, or excipient.Additionally, the present invention provides a pharmaceuticalcomposition comprising the Oxyntomodulin peptide analogue as definedabove, together with a pharmaceutically acceptable carrier, diluent, orexcipient and optionally other therapeutic ingredients.

Furthermore, the present invention provides a method of treatingnon-insulin-dependent (type 2) diabetes in a subject in need thereof,comprising administering to the subject in need thereof an effectiveamount of an Oxyntomodulin peptide analogue as defined above.

Additionally, the present invention provides a method of treatinginsulin-dependent (type 1) diabetes in a subject in need thereof,comprising administering to the subject in need thereof an effectiveamount of an Oxyntomodulin peptide analogue as defined above.

The present invention includes a method of treating obesity in a subjectin need thereof, comprising administering to the subject in need thereofan effective amount of an Oxyntomodulin peptide analogue as definedabove.

Furthermore, the present invention includes a method of treatingnon-insulin-dependent diabetes and obesity in a subject in need thereof,comprising administering to the subject in need thereof an effectiveamount of an Oxyntomodulin peptide analogue as defined above.

The present invention provides an Oxyntomodulin peptide analogue asdefined above for use as a medicament.

Additionally, the present invention provides an Oxyntomodulin peptideanalogue as defined above for use in the treatment ofnon-insulin-dependent diabetes.

Furthermore, the present invention provides an Oxyntomodulin peptideanalogue as defined above for use in the treatment of insulin-dependentdiabetes.

Furthermore, the present invention provides an Oxyntomodulin peptideanalogue as defined above for use in the treatment of obesity.

The present invention includes an Oxyntomodulin peptide analogue asdefined above for use in the treatment of non-insulin-dependent diabetesand obesity.

The present invention provides the use of an Oxyntomodulin peptideanalogue as defined above in the manufacture of a medicament for thetreatment of non-insulin-dependent diabetes.

Additionally, the present invention includes the use of an Oxyntomodulinpeptide analogue as defined above in the manufacture of a medicament forthe treatment of insulin-dependent diabetes.

Furthermore, the present invention provides the use of an Oxyntomodulinpeptide analogue as defined above in the manufacture of a medicament forthe treatment of obesity.

Furthermore, the present invention provides the use of an Oxyntomodulinpeptide analogue as defined above in the manufacture of a medicament forthe treatment of non-insulin-dependent diabetes and obesity.

The OXM peptide analogues of the present invention effectively bind toand activate both the GLP-1 receptor (GLP-1R) and glucagon receptor(GcgR).

It has also been found that the OXM peptide analogues of the presentinvention are more resistant to degradation by peptidases, in particulardipeptidyl peptidase IV than native human OXM. As a result, the OXMpeptide analogues of the present invention possess improved in vivostability versus native human OXM.

Various embodiments according to the present invention are capable ofcausing a reduction in food intake in overweight and obese subjects.

A particular advantage of the present invention is that the frequency ofside-effects, such as nausea, which is commonly associated with GLP-1based therapy, such as exenatide and liraglutide, is reduced oreliminated. The present invention therefore has reduced side-effectscompared to GLP-1 based therapy.

The OXM peptide analogues of the present invention also have superiorweight loss effect versus wild type human OXM.

Oxyntomodulin (OXM) is a weak co-agonist with full efficacy and balancedpotency at the hGLP-1R and hGcgR, with EC₅₀ values of about 6.7±2.7 nMand 4.1±1.7 nM, respectively in HEK293 cells stably overexpressing therespective receptors. The sequence of native human OXM is given below:

(SEQ ID NO: 4) His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile- Ala

The OXM peptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) atposition 39 is amidated is a fully efficacious and potent Oxyntomodulinpeptide analogue with an EC₅₀ of 60.0±2.97 nM and 6.58±0.87 nM againstthe hGcgR and hGLP-1R, respectively. The OXM peptide analogue of SEQ IDNO: 3 wherein the Cys(PEG20k) at position 39 is amidated has a balanceof in vitro functional activities that is ˜9-fold more selective for thehGLP-1R as compared to hGcgR. Comparable results are observed for thebinding affinities, Ki, where the OXM peptide analogue of SEQ ID NO: 3wherein the Cys(PEG20k) at position 39 is amidated is 3.6-fold moreselective for the hGLP-1R as compared to the hGcgR, with Ki values of1540±158 nM and 5500±350 nM, respectively.

The covalent attachment of one or more molecules of PEG to particularresidues of the OXM peptide analogue results in a PEGylated OXM peptideanalogue with an extended half-life and reduced rate of clearance, whencompared to that of the non-PEGylated OXM peptide analogue, and in vitropotency at the GLP-1R similar to that of native human OXM. Given thesmall size of the OXM peptide analogue and the relatively large size ofthe PEG molecule(s), it would be expected that the OXM peptide analogue,once PEGylated, would lose activity as a result of steric hindrance. Ithas been found, however, that if placed at the end of the Oxyntomodulinpeptide analogue rather than in the middle, the activity of the peptideanalogue is retained to a greater extent. Several substitutions in thesequence enhance potency, thereby offsetting the potency loss due toPEGylation while maintaining an appropriate ratio of activities at theGLP-1R and GcgR. Furthermore, it has been found that the presence of twoPEG molecules at the C-terminal end of the OXM peptide analogue ispreferable to a single PEG.

The sequences of the present invention contain the standard singleletter or three letter codes for the twenty naturally-occurring aminoacids. The other codes used are defined as follows:

-   -   d or D=the D isoform (nonnaturally occurring) of the respective        amino acid, e.g., D-Ser=D-Serine, dS=D-Serine    -   Aib=alpha amino isobutyric acid    -   1-Nal=1-naphthylalanine    -   PEG=polyethylene glycol    -   PEG20K=PEG molecule with average molecular weight of 20,000 Da

The term “PEG” as used herein means a polyethylene glycol molecule. Inits typical form, PEG is a linear polymer with terminal hydroxyl groupsand has the formula HO—CH₂CH₂—(CH₂CH₂O)n-CH₂CH₂—OH, where n is fromabout 8 to about 4000. Typically, n is not a discrete value butconstitutes a range with approximately Gaussian distribution around anaverage value. The terminal hydrogen may be substituted with a cappinggroup such as an alkyl or alkanol group. Preferably, PEG has at leastone hydroxy group, more preferably it is a terminal hydroxy group. Thishydroxy group is preferably attached to a linker moiety, which can reactwith the peptide to form a covalent linkage. Numerous derivatives of PEGexist in the art. (See, e.g., U.S. Pat. Nos. 5,445,090; 5,900,461;5,932,462; 6,436,386; 6,448,369; 6,437,025; 6,448,369; 6,495,659;6,515,100 and 6,514,491 and Zalipsky, S. Bioconjugate Chem. 6:150-165,1995). The PEG molecule covalently attached to the OXM peptide of thepresent invention may be approximately 10,000, 20,000, 30,000, or 40,000daltons average molecular weight. The PEG molecule is preferably 18,000to 22,000 daltons. More preferably, it is 19,000 to 21,000 Daltons. Mostpreferably it is 20,000 to 21,000 daltons. It is even more preferablyapproximately 20,000 daltons. PEGylation reagents may be linear orbranched molecules and may be present singularly or in tandem. ThePEGylated OXM peptide analogues of the present invention preferably havetandem PEG molecules attached to the C-terminus of the peptide. The PEGmolecules are preferably attached to the two cysteine residues at theC-terminal end of the peptide by an mPEG-20 kDa maleimide (Formula 1) oran mPEG-20 kDa iodoacetamide (Formula 2). In Formula 1 and Formula 2, nis 10 to 2500. Preferably, n is 350 to 600. More preferably, n is 425 to475.

In particular, the PEG molecules are preferably mPEG-20 kDa maleimide(CH₃O(CH₂CH₂O)_(n)—(CH₂)₃NHCO(CH₂)₂-maleimide) (NOF Sunbright ME-200MA)and are attached to the two cysteine residues at the C terminus of thepeptide. The most preferred Oxyntomodulin peptide analogue comprises theamino acid sequence of SEQ ID NO: 3, wherein the PEG molecules aremPEG-20 kDa maleimide (CH₃O(CH₂CH₂O)_(n)—(CH₂)₃NHCO(CH₂)₂-maleimide)(NOF Sunbright ME-200MA), and wherein the carboxyl group of thePEGylated Cys at position 39 is amidated (Formula 3). Formula 3 containsthe standard single letter amino acid code with exception of the boxareas where the structures for these amino acid residues have beenexpanded.

The term “PEGylation” as used herein means the covalent attachment ofone or more PEG molecules, as described above, to a molecule such as theOXM peptide analogues of the present invention.

“Insulinotropic activity” refers to the ability to stimulate insulinsecretion in response to elevated glucose levels, thereby causingglucose uptake by cells and decreased plasma glucose levels.Insulinotropic activity can be assessed by methods known in the art,including in vitro experiments that measure insulin secretion byinsulinoma cell lines or islets or in vivo experiments such asintravenous glucose tolerance test (IVGTT), intraperitoneal glucosetolerance test (IPGTT), and oral glucose tolerance test (OGTT).Insulinotropic activity is routinely measured in humans by measuringplasma insulin or C-peptide levels. The OXM peptide analogues of thepresent invention possess robust insulinotropic activity.

“In vitro potency” as used herein is the measure of the ability of theOXM peptide analogue to activate the GLP-1R or the GcgR in a cell-basedassay. In vitro potency is expressed as the “EC₅₀”, which is theeffective concentration of compound that results in a half maximalincrease in the measured response (in this case, cyclic AMP production)in a dose-response experiment.

The term “plasma half-life” refers to the time required for half of therelevant molecules to be cleared from the plasma. An alternatively usedterm is “elimination half-life.” The term “extended” or “longer” used inthe context of plasma half-life or elimination half-life indicates thereis a significant increase in the half-life of a PEGylated OXM peptideanalogue relative to that of the reference molecule (e.g., thenon-PEGylated form of the peptide or the native peptide) as determinedunder comparable conditions. The half-life of native OXM in monkeys, forexample, is expected to be less than 1 hr. The PEGylated OXM peptideanalogues of the present invention have an elimination half-life of atleast 24 hr in monkey, and preferably at least 48 hours. The half-lifereported herein is the elimination half-life, which corresponds to theterminal log-linear rate of elimination. The person skilled in the artappreciates that half-life is a derived parameter that changes as afunction of both clearance and volume of distribution.

The term “long-acting GLP-1R agonist” as used herein, refers to a GLP-1peptide analogue covalently attached to one or more molecules ofpolyethylene glycol (PEG). PEGylated GLP-1 compounds are disclosed inU.S. Pat. No. 7,557,183.

Clearance is the measure of the body's ability to eliminate a drug fromcirculation. As clearance decreases due, for example, to modificationsto a drug, half-life would be expected to increase. However, thisreciprocal relationship is exact only when there is no change in thevolume of distribution. A useful approximate relationship between theterminal log-linear half-life (t_(1/2)), clearance (C), and volume ofdistribution (V) is given by the equation: t_(1/2)≈0.693 (V/C).Clearance does not indicate how much drug is being removed but, rather,the volume of biological fluid such as blood or plasma that would haveto be completely freed of drug to account for the elimination. Clearanceis expressed as a volume per unit of time. The PEGylated OXM peptideanalogues of the present invention preferably have a clearance value of200 ml/h/kg or less in monkeys, more preferably 180, 150, 120, 100, 80,60 ml/h/kg or less and most preferably 50, 40 or 20 ml/h/kg or less.

The OXM peptide analogues of the present invention typically will beadministered parenterally. Parenteral administration includes, forexample, systemic administration, such as by intramuscular, intravenous,subcutaneous, intradermal, or intraperitoneal injection. The peptideanalogue is administered to the subject in conjunction with anacceptable pharmaceutical carrier, diluent, or excipient as part of apharmaceutical composition for treating non-insulin dependent (type 2)diabetes mellitus, NIDDM, or the related disorders discussed below. Thepharmaceutical composition can be a solution or a suspension of the OXMpeptide analogue, such as one in which the OXM peptide analogue iscomplexed with a divalent metal cation such as zinc. The peptideanalogue may also be formulated in a solid formulation such as bylyophilisation or spray-drying, which is then reconstituted in asuitable diluent solution prior to administration. Suitablepharmaceutical carriers may contain inert ingredients which do notinteract with the peptide or peptide derivative. Suitable pharmaceuticalcarriers for parenteral administration include, for example, sterilewater, physiological saline, bacteriostatic saline (saline containingabout 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank'ssolution, Ringer's-lactate and the like. Some examples of suitableexcipients include lactose, dextrose, sucrose, trehalose, sorbitol, andmannitol and preservatives such as phenol and m-cresol.

Standard pharmaceutical formulation techniques, such as those describedin Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton,Pa.), may be employed. The OXM peptide analogues of the presentinvention may alternatively be formulated for administration through thebuccal, oral, transdermal, nasal, or pulmonary route. The OXM peptideanalogues of the invention may be formulated for extended release suchthat blood plasma levels are maintained in the efficacious range forextended time periods after administration.

The OXM peptide analogues of the present invention may be employed totreat diabetes, specifically type 2 diabetes (non-insulin dependentdiabetes mellitus or NIDDM). Additional subjects who may benefit fromtreatment with the OXM peptide analogues of the present inventioninclude those with impaired glucose tolerance or impaired fastingglucose, subjects whose body weight is about 25% or more above normalbody weight for the subject's height and body build, subjects having oneor more parents with NIDDM, subjects who have had gestational diabetes,and subjects with metabolic disorders such as those resulting fromdecreased endogenous insulin secretion. The OXM peptide analogue may beused to prevent subjects with impaired glucose tolerance from proceedingto develop type 2 diabetes, prevent pancreatic β-cell deterioration,induce β-cell proliferation, improve β-cell function, activate dormantβ-cells, promote differentiation of cells into β-cells, stimulate β-cellreplication, and inhibit β-cell apoptosis. Other diseases and conditionsthat may be treated or prevented using compounds of the invention inmethods of the invention include: Maturity-Onset Diabetes of the Young(MODY) (Herman, et al., Diabetes 43:40, 1994); Latent AutoimmuneDiabetes Adult (LADA) (Zimmet, et al., Diabetes Med. 11:299, 1994);impaired glucose tolerance (IGT) (Expert Committee on Classification ofDiabetes Mellitus, Diabetes Care 22 (Supp. 1):S5, 1999); impairedfasting glucose (IFG) (Charles, et al., Diabetes 40:796, 1991);gestational diabetes (Metzger, Diabetes, 40:197, 1991); metabolicsyndrome X, dyslipidemia, hyperglycemia, hyperinsulinemia,hypertriglyceridemia, and insulin resistance.

The OXM peptide analogues of the invention may also be used in methodsof the invention to treat secondary causes of diabetes (Expert Committeeon Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1):S5,1999). Such secondary causes include glucocorticoid excess, growthhormone excess, pheochromocytoma, and drug-induced diabetes. Drugs thatmay induce diabetes include, but are not limited to, pyriminil,nicotinic acid, glucocorticoids, phenyloin, thyroid hormone,β-adrenergic agents, α-interferon and drugs used to treat HIV infection.

The OXM peptide analogues of the present invention may be effective inthe suppression of food intake and the treatment of obesity.

An “effective amount” of an OXM peptide analogue is the quantity thatresults in a desired therapeutic and/or prophylactic effect withoutcausing unacceptable side effects when administered to a subject. A“desired therapeutic effect” includes one or more of the following: 1)an amelioration of the symptom(s) associated with the disease orcondition; 2) a delay in the onset of symptoms associated with thedisease or condition; 3) increased longevity compared with the absenceof the treatment; and 4) greater quality of life compared with theabsence of the treatment. For example, an “effective amount” of an OXMpeptide analogue for the treatment of NIDDM is the quantity that wouldresult in greater control of blood glucose concentration than in theabsence of treatment, thereby resulting in a delay in the onset ofdiabetic complications such as retinopathy, neuropathy, or kidneydisease. An “effective amount” of an OXM peptide analogue for theprevention of NIDDM, for example in subjects with impaired glucosetolerance or impaired fasting glucose, is the quantity that would delay,compared with the absence of treatment, the onset of elevated bloodglucose levels that require treatment with anti-hyperglycemic drugs suchas sulfonylureas, thiazolidinediones, insulin, and/or bisguanidines.

An “effective amount” of an OXM peptide analogue administered to asubject will also depend on the type and severity of the disease and onthe characteristics of the subject, such as general health, age, sex,body weight and tolerance to drugs. The dose of OXM peptide analogueeffective to normalize a subject's blood glucose will depend on a numberof factors, among which are included, without limitation, the subject'ssex, weight and age, the severity of inability to regulate bloodglucose, the route of administration and bioavailability, thepharmacokinetic profile of the peptide, the potency, and theformulation.

A typical once weekly dose for the PEGylated OXM peptide analogues ofthe present invention preferably will range from about 0.1 mg to about1000 mg (total weight of the conjugate). More preferably, the onceweekly dose will range from about 1 mg to about 100 mg, or about 1 mg toabout 30 mg. Most preferably, the once weekly dose will range from about5 mg to about 30 mg, or about 1 mg to about 5 mg.

A “subject” is a mammal, preferably a human, but can also be an animal,including companion animals (e.g., dogs, cats, and the like), farmanimals (e.g., cows, sheep, pigs, horses, and the like) and laboratoryanimals (e.g., rats, mice, guinea pigs, and the like).

Various preferred features and embodiments of the present invention willnow be described only by way of the examples.

EXAMPLE 1 Peptide Synthesis

The peptide analogue according to SEQ ID NO: 1 and SEQ ID NO: 2 of thepresent invention is generated by solid-phase peptide synthesis on aProtein Technologies Inc. Symphony or Applied Biosystems 433A automatedpeptide synthesizer. Synthesis is performed on Fmoc-Rink amidepolystyrene resin (Rapp Polymere Tubingen, Germany) with substitutionapproximately 0.7 mmol/g. The synthesis is performed using the Fmocmain-chain protecting group strategy. Amino acid side-chain derivativesused are: Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Trt), Gln(Trt), Glu(OtBu),His(Trt), Lys(Boc), Ser(OtBu), Thr(OtBu), Trp(Boc) and Tyr(OtBu).Coupling is carried out with approximately 10 equivalents of amino acidactivated with diisopropylcarbodiimide (DIC) and hydroxybenzotriazole(HOBt) (1:1:1 molar ratio) in dimethylformamide (DMF) orN-methylpyrrolidinone (NMP). Coupling is carried out for 45 to 90minutes at room temperature.

Concomitant cleavage from the resin and side chain protecting groupremoval are carried out in a solution containing trifluoroacetic acid(TFA): triisopropylsilane: 3,6-dioxa-1,8-octane-dithiol: methanol:anisole 90:4:2:2:2 (v/v) for 1.5 to 2 hours at room temperature. Thesolution is filtered and concentrated to <2 mL, and peptides areprecipitated with cold diethyl ether, redissolved in 30-40 mL of 10%acetonitrile and purified on a C₁₈ reversed-phase high performanceliquid chromatography (HPLC) column (typically a Waters SymmetryPrep 7um, 19×300 mm) at a flow rate of 12-15 mL/min. Samples are eluted with atwo-stage linear AB gradient of 0 to 25% B over 20 minutes followed by25 to 75% B over 100 minutes where A=0.05% TFA/water and B=0.05%TFA/acetonitrile. Product generally elutes at 30-35% acetonitrile.Peptide purity and molecular weight is confirmed on an Agilent 1100Series liquid chromatography-mass spectrometry (LC-MS) system with asingle quadrupole MS detector. Analytical HPLC separation is done on aZorbax Eclipse XDB-C8, 5 micron, 4.6 mm i.d.×15 cm column with a linearAB gradient of 6 to 60% B over 15 minutes in which A=0.05% TFA/H₂O andB=0.05% TFA/acetonitrile and the flow rate is 1 ml/min. The peptideanalogue is purified to >95% purity and is confirmed to have molecularweight corresponding to the calculated value within 1 atomic mass unit(amu).

EXAMPLE 2 PEGylation of Peptide Containing Two Cys Residues withmPEG-MAL-20 kDa

The lyophilized peptide analogue (SEQ ID NO: 2) generated according toExample 1 is weighed out (typically 30-50 mg). A 2.1 fold molarequivalent of mPEG-20 kDa maleimide(CH₃O(CH₂CH₂O)_(n)—(CH₂)₃NHCO(CH₂)₂-maleimide) (NOF Sunbright ME-200MA)is weighed out and combined with the peptide. The reactants aredissolved in a 50/50 (v/v) water/acetonitrile mixture to a peptideconcentration of approximately 20 mg/mL. The peptide analogue solutionis diluted two-fold with 100 mM ammonium acetate, 10 mMethylenediaminetetraacetic acid (EDTA), pH 7. The resultant mixture isthen stirred at room temperature. The reaction mixture is monitored byanalytical reversed phase HPLC (analytical HPLC separation is done on aWaters SymmetryShield C18, 3.5 micron, 4.6 mm i.d.×10 cm column at 50°C. with a two-stage linear AB gradient of 0 to 30% B over 5 minutes and30 to 90% B over the subsequent 30 min in which A=0.05% TFA/H₂O andB=0.05% TFA/acetonitrile and the flow rate is 1 ml/min), and typicallyafter 1-2 hr reaction time, shows almost complete disappearance of thepeptide peak. Two peaks due to mono- and di-PEGylated peptide appearwith the di-PEGylated peptide typically constituting 90-95% of the totalpeak area. The sample is then diluted to about 20 mL with water andpurified as in Example 1 with a two-stage linear AB gradient of 0 to 30%B over 20 min followed by 30 to 80% B over 100 min. Product generallyelutes at 35-40% acetonitrile. The purified peptide is quantitated byultraviolet (UV) absorbance at 280 nm using a calculated molarextinction coefficient based on the peptide sequence. Yield afterpurification is in the range of 70 to 80% based on the amount ofstarting peptide.

EXAMPLE 3 Glucagon Receptor (hGcgR) Binding Assay

The Glucagon receptor binding assay utilizes cloned human glucagonreceptor (hGcgR) (Lok S, Kuijper J L, Jelinek L J, Kramer J M, WhitmoreT E, Sprecher C A, Mathewes S, Grant F J, Biggs S H, Rosenberg G B, etal. Gene 140 (2), 203-209 (1994)) isolated from 293HEK membranes. ThehGlucR cDNA is subcloned into the expression plasmid phD(Trans-activated expression of fully gamma-carboxylated recombinanthuman protein C, an antithrombotic factor. Grinnell, B. W., Berg, D. T.,Walls, J. and Yan, S. B. Bio/Technology 5: 1189-1192 (1987)). Thisplasmid DNA is transfected into 293HEK cells and selected with 200 μg/mlHygromycin.

Crude plasma membranes are prepared using cells from suspension culture.The cells are lysed on ice in hypotonic buffer containing 25 mM TrisHCl, pH 7.5, 1 mM MgCl₂, DNAse1, 20 ug/ml, and Roche Complete Inhibitorswithout EDTA. The cell suspension is homogenized with a glass douncehomogenizer using a Teflon pestle for 25 strokes. The homogenate iscentrifuged at 4° C. at 1800×g for 15 minutes. The supernatant iscollected and the pellet is resuspended in hypotonic buffer andrehomogenized. The mixture is centrifuged at 1800×g for 15 minutes. Thesecond supernatant is combined with the first supernate. The combinedsupernatants are centrifuged at 1800×g for 15 minutes to clarify. Theclarified supernatant is transferred to high speed tubes and centrifugedat 25000×g for 30 minutes at 4° C. The membrane pellet is resuspended inhomogenization buffer and stored as frozen aliquots at −80° C. freezeruntil use.

Glucagon is radioiodinated by ¹²⁵I-lactoperoxidase procedure andpurified by reversed phase HPLC at Perkin-Elmer/NEN (NEX207). Thespecific activity is about 2200 Ci/mmol K_(D) determination is performedby homologous competition instead of saturation binding due to highpropanol content in the ¹²⁵I-labelled glucagon material. The K_(D) isestimated to be 2.62 nM and is used to calculate Ki values for allcompounds tested.

The receptor binding assay is carried out using a ScintillationProximity Assay (SPA) with wheat germ agglutinin (WGA) beads previouslyblocked with 1% fatty acid free bovine serum albumin (BSA). The bindingbuffer contains 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), pH 7.4, 2.5 mM CaCl₂, 1 mM MgCl₂, 0.1% fatty acid free BSA,0.003% Tween20, and Roche Complete Inhibitors without EDTA. Glucagon isdissolved in 0.01 N HCl at 1 mg/ml and immediately frozen at −80° C. in30 μl aliquots. Glucagon aliquots are diluted and used in the bindingassay within 1 hr. The OXM peptide analogue is dissolved in phosphatebuffered saline (PBS) and serially diluted in binding buffer. Next, 10μl diluted compounds or PBS is transferred into Corning 3632 clearbottom assay plates containing 40 p. 1 assay binding buffer or coldglucagon (non-specific binding (NSB) at 1 μM final). Then, 90 μlmembranes (3 μg/well), 50 μl ¹²⁵I-Glucagon (0.15 nM final concentrationin reaction), and 50 μl of WGA beads (150 μg/well) are added. Plates aresealed, mixed end over end, and read with a MicroBeta scintillationcounter after 12 hr of settling time at room temperature.

Results are calculated as a percent of specific ¹²⁵I-glucagon binding inthe presence of compound. The absolute IC₅₀ concentration of compound isderived by non-linear regression of percent specific binding of¹²⁵I-glucagon vs. the concentration of compound added. The IC₅₀ dose isconverted to Ki using the Cheng-Prusoff equation (Cheng Y., Prusoff W.H., Biochem. Pharmacol. 22, 3099-3108, 1973). The Ki of the OXM peptideanalogue of SEQ ID NO: 3 wherein the Cys(PEG20k) at position 39 isamidated was 5500±350 nM for hGcgR binding.

EXAMPLE 4 Glucagon-Like-Peptide 1 (hGLP-1-R) Receptor Binding Assay

The GLP-1 receptor binding assay uses cloned human glucagon-like peptide1 receptor (hGLP-1R) (Graziano M P, Hey P J, Borkowski D, Chicchi G G,Strader C D, Biochem Biophys Res Commun. 1993 Oct. 15; 196(1):141-6)isolated from 293HEK membranes. The hGLP-1R cDNA is subcloned into theexpression plasmid phD (Trans-activated expression of fullygamma-carboxylated recombinant human protein C, an antithromboticfactor. Grinnell, B. W., Berg, D. T., Walls, J. and Yan, S. B.Bio/Technology 5: 1189-1192 (1987)). This plasmid DNA is transfectedinto 293HEK cells and selected with 200 μg/ml Hygromycin.

Crude plasma membranes are prepared using cells from suspension culture.The cells are lysed on ice in hypotonic buffer containing 25 mM TrisHCl, pH 7.5, 1 mM MgCl₂, DNAse1, 20 ug/ml, and Roche Complete Inhibitorswithout EDTA. The cell suspension is homogenized with a glass douncehomogenizer using a Teflon pestle for 25 strokes. The homogenate iscentrifuged at 4° C. at 1800×g for 15 minutes. The supernatant iscollected and the pellet is resuspended in hypotonic buffer andrehomogenized. The mixture is centrifuged at 1800×g for 15 minutes. Thesecond supernatant is combined with the first supernatant. The combinedsupernatants are centrifuged at 1800×g for 15 minutes to clarify. Theclarified supernatant is transferred to high speed tubes and centrifugedat 25000×g for 30 minutes at 4° C. The membrane pellet is resuspended inhomogenization buffer and stored as frozen aliquots at −80° C. freezeruntil use.

Glucagon-like peptide 1 (GLP-1) is radioiodinated by the¹²⁵I-lactoperoxidase procedure and purified by reversed phase HPLC atPerkin-Elmer/NEN (NEX308). The specific activity is about 2200 Ci/mmolK_(D) determination is performed by homologous competition instead ofsaturation binding due to high propanol content in the ¹²⁵I-GLP-1material. The K_(D) is estimated to be 0.96 nM and is used to calculateKi values for all compounds tested.

The receptor binding assay is carried out using a ScintillationProximity Assay (SPA) with wheat germ agglutinin (WGA) beads previouslyblocked with 1% fatty acid free BSA (ICN). The binding buffer contains25 mM HEPES, pH 7.4, 2.5 mM CaCl₂, 1 mM MgCl₂, 0.1% fatty acid free BSA,0.003% Tween20, and Roche Complete Inhibitors without EDTA. GLP-1 isdissolved in PBS at 1 mg/ml and immediately frozen at −80° C. in 30 μlaliquots. GLP-1 aliquots are diluted and used in binding assays within 1hr. The OXM peptide analogue is dissolved in PBS and serially diluted inbinding buffer. Next, 10 μl diluted compounds or PBS is transferred intoCorning 3632 clear bottom assay plates containing 40 μl assay bindingbuffer or cold GLP-1 (NSB at 1 μM final). Then, 90 μl membranes (1μg/well), 50 μl ¹²⁵I-GLP-1 (0.15 nM final concentration in reaction),and 50 μl of WGA beads (150 μg/well) are added. Plates are sealed, mixedend over end, and read with a MicroBeta scintillation counter after 12hr of settling time at room temperature.

Results are calculated as a percent of specific ¹²⁵I-GLP-1 binding inthe presence of compound. The Absolute IC₅₀ concentration of compound isderived by non-linear regression of percent specific binding of¹²⁵I-GLP-1 vs. the concentration of compound added. The IC₅₀concentration is converted to Ki using the Cheng-Prusoff equation (ChengY., Prusoff W. H., Biochem. Pharmacol. 22, 3099-3108, 1973). The Ki ofthe OXM peptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) atposition 39 is amidated was 1540±158 nM for hGLP-1R binding.

EXAMPLE 5 Glucagon Receptor-Stimulated cAMP Functional Assay

The Glucagon stimulated cAMP functional assay uses the same cloned hGcgRexpressing cell line as used for the hGcgR binding assay described abovein Example 3. Cells are stimulated with the OXM peptide analogue, andthe cAMP generated within the cell is quantitated using an AmplifiedLuminescent Proximity Homogeneous Assay (Alpha Screen) from Perkin Elmer(6760625R). Briefly, cAMP induced within the cell competes for bindingof biotinylated cAMP from the kit to a coated anti-cAMP antibodyAcceptor bead and a strepavidin coated Donor bead. As the cAMP levelwithin the cell increases, a disruption of the Acceptorbead-biotinylated cAMP-Donor bead complex occurs and decreases thesignal which is observed.

The hGcgR-HEK293 cells are harvested from sub-confluent tissue culturedishes with Enzyme-Free Cell Dissociation Solution, (Specialty Media5-004-B). The cells are pelleted at low speed and washed 3 times withassay buffer [25 mM HEPES in Hank's buffered salt solution (HBSS)-withMg and Ca (GIBCO, 14025-092) with 0.1% Fatty Acid Free BSA] then dilutedto a final concentration of 125,000 cells per ml. Biotinylated cAMP fromthe Alpha Screen kit is added to the diluted cells at a finalconcentration of 1 unit/0.04 ml. A phosphodiesterase inhibitor, IBMX(250 mM in dimethyl sulfoxide (DMSO)), is also added to the dilutedcells to a final concentration of 500 uM. Glucagon is initiallydissolved in 0.01 N HCl at 1 mg/ml and immediately frozen at −80° C.Upon thawing, the glucagon should be used within 1 hr. The glucagon,cAMP standard, and OXM peptide analogue are serially diluted into Assaybuffer to a 6× final concentration. The functional assay is performed in96 well, low-volume, white, polystyrene Costar Plates (3688). Thereaction starts by adding 0.01 ml of the diluted OXM peptide analogue,glucagon, or cAMP into 0.04 ml of the cell mixture. After one hour atroom temperature, the reaction is stopped by the addition of 0.03 ml ofLysis Buffer [10 mM HEPES, pH 7.4, 1% NP40, and 0.01% fatty acid freeBSA containing 1 unit each/0.03 ml of Acceptor and Donor beads from theAlpha Screen Kit]. Addition of the lysis buffer is performed in the darkto prevent bleaching of the detection beads. The plates are wrapped infoil, gently shaken for 1 minute then left to equilibrate overnight atroom temperature. The plates are read on a Perkin-Elmer Envisioninstrument. The Alpha screen units are converted into pmoles cAMPgenerated per well based upon the cAMP standard curve. The pmoles cAMPgenerated in each well is converted to a percent of the maximal responseobserved with the glucagon control. An EC₅₀ value is derived bynon-linear regression analysis using the percent maximal response vs.the concentration of peptide added. The OXM peptide analogue of SEQ IDNO: 3 wherein the Cys(PEG20k) at position 39 is amidated, like wild typeOXM, was fully efficacious and potent at hGcgR with EC₅₀ of 60.0±2.97nM.

EXAMPLE 6 Glucagon-Like-Peptide 1 (hGLP-1) Receptor-Stimulated cAMPFunctional Assay

The GLP-1 stimulated cAMP functional assay uses the same cloned hGLP-1Rexpressing cell line as used for the hGLP-1R binding assay describedabove in Example 4. Cells are stimulated with the OXM peptide analogue,and the cAMP generated within the cell is quantitated using an AmplifiedLuminescent Proximity Homogeneous Assay (Alpha Screen) from Perkin Elmer(6760625R). Briefly, cAMP induced within the cell competes for bindingof biotinylated cAMP from the kit to a coated anti-cAMP antibodyAcceptor bead and a strepavidin coated Donor bead. As the cAMP levelwithin the cell increases, a disruption of the Acceptorbead-biotinylated cAMP-Donor bead complex occurs and decreases thesignal which is observed.

The hGLP-1R-HEK293 cells are harvested from sub-confluent tissue culturedishes with Enzyme-Free Cell Dissociation Solution, (Specialty Media5-004-B). The cells are pelleted at low speed and washed 3 times withassay buffer [25 mM HEPES in HBSS-with Mg and Ca (GIBCO, 14025-092) with0.1% Fatty Acid Free BSA] then diluted to a final concentration of125,000 cells per ml. Biotinylated cAMP from the Alpha Screen kit isadded to the diluted cells at a final concentration of 1 unit/0.04 ml. Aphosphodiesterase inhibitor, IBMX (250 mM in DMSO), is also added to thediluted cells to a final concentration of 500 μM. GLP-1 is stored at 1mg/ml in PBS as frozen aliquots at −80° C. The GLP-1, cAMP standard, andOXM peptide analogue are serially diluted into Assay buffer to a 6×final concentration. The functional assay is performed in 96 well, lowvolume, white, polystyrene Costar Plates (3688). The reaction starts byadding 0.01 ml of the diluted OXM peptide analogue, GLP-1, or cAMP into0.04 ml of the cell mixture. After 1 hr at room temperature, thereaction is stopped by the addition of 0.03 ml of Lysis Buffer [10 mMHEPES, pH 7.4, 1% NP40, and 0.01% fatty acid free BSA containing 1 uniteach/0.03 ml of Acceptor and Donor beads from the Alpha Screen Kit].Addition of the lysis buffer is performed in the dark to preventbleaching of the detection beads. The plates are wrapped in foil, gentlyshaken for 1 minute then left to equilibrate overnight at roomtemperature. The plates are read on a Perkin-Elmer Envision instrument.The Alpha screen units are converted into pmoles cAMP generated per wellbased upon the cAMP standard curve. The pmoles cAMP generated in eachwell is converted to a percent of the maximal response observed with theGLP-1 control. An EC₅₀ value is derived by non-linear regressionanalysis using the percent maximal response vs. the concentration ofpeptide added. The OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated, like wild type OXM, was fullyefficacious and potent at hGLP-1R with EC₅₀ of 6.58±0.87 nM.

EXAMPLE 7 Effects on Food Intake, Body Weight and Body Composition inDiet-Induced Obese (DIO) Mice

Three to four months old male diet-induced obese (DIO) mice areindividually housed in a temperature-controlled (24° C.) facility with a12 hour light/dark cycle (lights on 22:00), and have free access to foodand water. After 2 weeks acclimation to the facility, mice arerandomized to treatment groups (n=8-10/group), each group having similarmean body weight and fat mass. Mice are subcutaneously (sc) injectedwith vehicle solution and weighed for 2 days to acclimate them to theprocedures.

Vehicle or OXM peptide analogue (dose range 7.5-30 nmole/kg) dissolvedin vehicle is administered by sc injection to ad libitum fed DIO mice30-90 minutes prior to the onset of the dark cycle every 3 days for 2weeks (two repeated studies). Body weight and the weight of food plusthe hopper are measured at the same time. Food consumed in thepreceeding 24 hours is calculated by subtracting current weight of foodplus the hopper from that of the previous day. Absolute changes in bodyweight are calculated by subtracting the body weight of the animal priorto the first injection. On days 1 and 14 total fat mass is measured bynuclear magnetic resonance (NMR) using an Echo Medical System (Houston,Tex.) instrument. Fat free mass is calculated by subtracting fat massfrom total body weight.

Study 1

The OXM peptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) atposition 39 is amidated is administered by subcutaneous injection to 4months old male diet-induced obese (DIO) mice. The OXM peptide analogueis injected once every 3 days for 2 weeks at doses of 7.5, 15 and 30nmole/kg and compared to vehicle treated mice and positive control (7.5nmole/kg of a long-acting GLP-1R agonist injected every 3 days) treatedanimals.

Treatment with the OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated produced a dose-dependentreduction in food intake and body weight. At the end of 2-week studyperiod, cumulative food intake in the 15 and 30 nmole/kg groups wasreduced by 21% and 27%, respectively when compared to the vehicle group.Cumulative weight loss of the 7.5 nmole/kg group was similar to thatobserved with the positive control, which is about 6% reduction whencompared to the vehicle group. Vehicle controlled cumulative weight lossof the 15 and 30 nmole/kg groups were 10% and 15% respectively. Bodycomposition analysis showed that the weight loss was primarily due toloss of fat mass (Table 1).

TABLE 1 Weight change in DIO mice over a 14-day treatment period (mean ±SEM; n = 8) Dose of OXM peptide analogue of SEQ ID NO: 3 wherein theOverall weight loss Fat mass loss (g fat Cys(PEG20k) at position 39 (gweight change for Total food intake weight change for 14 is amidated(nmole/kg) 14 days) (g total for 14 days) days) 0 (Vehicle) 0.7 ± 0.239.6 ± 0.7  0.4 ± 0.2 7.5 −1.7 ± 0.3* 36.5 ± 1.2* −1.1 ± 0.3* 15 −3.5 ±0.3* 31.4 ± 0.9* −2.7 ± 0.2* 30 −5.6 ± 0.4* 29.0 ± 1.3* −4.0 ± 0.3*

Study 2

The OXM peptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) atposition 39 is amidated (7.5 or 22.5 nmole/kg) and positive control (7.5or 22.5 nmole/kg of a long-acting GLP-1R agonist) are administered every3 days by subcutaneous injection to 4 months old male diet-induced obese(DIO) C57BL/6 for 2 weeks.

All treated groups exhibited significantly decreased cumulative foodintake relative to vehicle controls. The OXM peptide analogue andpositive control decreased body weight to similar degrees, about 5% and10% for the low and high doses, respectively. Body composition analysisconfirmed that weight loss associated with the OXM peptide analogue andpositive control is primarily due to loss of fat mass (Table 2).

TABLE 2 Weight change in DIO over a 14-day treatment period (mean ± SEM;n = 10) Dose of OXM peptide analogue of SEQ ID NO: 3 wherein the Overallweight loss Fat mass loss (g fat Cys(PEG20k) at position 39 (g weightchange for Total food intake weight change for 14 is amidated (nmole/kg)14 days) (g total for 14 days) days) 0 (Vehicle) 0.1 ± 0.2 38.8 ± 0.5 0.0 ± 0.2 7.5 −2.5 ± 0.3* 33.3 ± 0.6* −1.6 ± 0.2* 22.5 −5.2 ± 0.2* 28.7± 0.6* −3.8 ± 0.2* These data show that the OXM peptide analogue of SEQID NO: 3 wherein the Cys(PEG20k) at position 39 is amidated decreasedcumulative food intake and body weight in two repeated 14 day DIO mousestudies, compared to vehicle-treated mice. Reduced body weight wasprimarily due to reduction in fat mass. *p < 0.05 versus vehicle(Dunnett's test)

EXAMPLE 8 Effects on Blood Glucose Excursion During an IntraperitonealGlucose Tolerance Test or an Oral Glucose Tolerance Test after 2-WeekTreatment in DIO Mice

Fifty-six hours after the fifth injection as described in Example 7 inDIO mice, mice are fasted for 16 hours prior to the start of the glucosetolerance test. At time 0, animals are given 2 g/kg dextrose by oralgavage (Table 3, Study 1) or intraperitoneal (IP) injection (Table 4,Study 2). Blood is collected by tail vein bleeding at 0, 15, 30, 60 and120 minutes after glucose challenge. Glucose concentration is measuredby glucometer. All doses of the OXM peptide analogue of SEQ ID NO: 3wherein the Cys(PEG20k) at position 39 is amidated as well as thepositive control significantly lowered the blood glucose at all timepoints measured before and after the oral glucose challenge whencompared to the vehicle-treated controls.

TABLE 3 Effects of OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated on the blood glucose excursionfollowing administration of an oral glucose load Dose of OXM peptideanalogue of SEQ ID NO: 3 wherein the Cys(PEG20k) at position 39 GlucoseAUC (mg*min/dL) is amidated (nmole/kg) MEAN SEM 0 (Vehicle) 24786  11277.5 13802* 264 15 12668* 209 30 13056* 293 Data given as area under theglucose curve (= integrated values from t + 0 to 120 min) (n = 7) Thesedata show that the OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated significantly reduced the bloodglucose excursion following an oral glucose load. Statisticalsignificance evaluated by Dunnett's test. (*p < 0.05 vs. vehicle)

TABLE 4 Effects of OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated on the blood glucose excursionfollowing an intraperitoneal (ip) glucose load Dose of OXM peptideanalogue of SEQ ID NO: 3 wherein the Cys(PEG20k) at position 39 GlucoseAUC (mg*min/dL) is amidated (nmole/kg) MEAN SEM 0 (Vehicle) 32148  8947.5 19198* 2521 22.5 15720* 1054 Data given as area under the glucosecurve (= integrated values from t + 0 to 120 min) (n = 6) These datashow that the OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated significantly reduced the bloodglucose excursion following an intraperitoneal (ip) glucose load.Statistical significance evaluated by Dunnett's test. (*p < 0.05 vs.vehicle)

EXAMPLE 9 Effects on Blood Glucose Excursion During an IntraperitonealGlucose Tolerance Test in Lean Mice

Nine week old male C57BL/6 mice are used in the study Animals arerandomized into groups based on fed body weight. Animals are injectedwith vehicle or OXM peptide analogue (dose 5.0-15.0 nmole/kg) 16 hoursprior to the start of the test. Food is removed at the time of injectionof peptide or vehicle. At time 0, animals are given 2 g/kg dextrose byIP injection. Blood is collected by tail vein bleeding at 0, 3, 6, 12and 30 minutes after glucose challenge. Glucose concentration ismeasured by glucometer. Insulin is measure by Mesoscale.

Both doses of the OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated significantly lowered bloodglucose excursion (Table 5) and significantly increased plasma insulinconcentrations (Table 6) when compared to the vehicle-treated controls.

TABLE 5 Effects of OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated on blood glucose excursionfollowing an intraperitoneal (ip) glucose tolerance test in lean miceDose of OXM peptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) atposition 39 Glucose AUC (mg*min/dL) is amidated (nmole/kg) MEAN SEMVehicle 9816  532 5 6287* 720 15 6881* 331 Data given as area under theglucose curve (= integrated values from t + 0 to 30 min) (n = 6) Thesedata show that the OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated significantly reduced the bloodglucose excursion following an intraperitoneal (ip) glucose tolerancetest in lean mice. Statistical significance evaluated by Dunnett's test.(*p < 0.05 vs. vehicle)

TABLE 6 Effects of OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated on plasma insulin concentrationfollowing an intraperitoneal (ip) glucose tolerance test in lean miceDose of OXM peptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) atposition 39 Insulin AUC (ng*min/mL) is amidated (nmole/kg) MEAN SEM 0(Vehicle) 9.44 0.80 5 33.29* 5.88 15 58.32* 8.86 Data given as areaunder the insulin curve (= integrated plasma insulin values from t + 0to 30 min) (n = 6) These data show that the OXM peptide analogue of SEQID NO: 3 wherein the Cys(PEG20k) at position 39 is amidatedsignificantly increased plasma insulin AUC following an intraperitoneal(ip) glucose tolerance test in lean mice. Statistical significanceevaluated by Dunnett's test. (*p < 0.05 vs. vehicle)

EXAMPLE 10 Effects on Blood Glucose Excursion During an Oral GlucoseTolerance Test (OGTT) or an Intraperitoneal (IP) Glucose Tolerance Test(IPGTT) in Obese (ob/ob) Mice

Two to three months old male ob/ob mice are individually housed in atemperature-controlled (24° C.) facility with a 12 hour light/dark cycle(lights on 2200 hours), and have free access to food and water. After atleast 2 weeks of acclimation to the facility, 3-hours fasting bloodglucose is measured by tail vein bleeding at 9 AM. Mice with bloodglucose under 180 mg/dL are not used. Remaining animals are randomizedto treatment groups (N=7/group), each group having similar average bloodglucose level. The mice are given access to food until the time ofinjection. Animals are injected with vehicle, 7.5 nmole/kg or 15nmole/kg OXM peptide analogue at 4 PM of the same day. Food is removedat the time of injection. An OGTT (Table 7) or IPGTT (Table 8) isperformed 16 hours after the peptide injection. At time 0, animals aregiven 2 g/kg dextrose by oral gavage (Table 7) or intraperitonealinjection (Table 8). Blood is collected by tail vein bleeding at 0, 15,30, 60 and 120 minutes after glucose challenge. Blood glucoseconcentration is measured by glucometer. A single injection of the OXMpeptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) at position 39is amidated normalized blood glucose in ob/ob mice. Glucose levels atall time points measured after glucose challenge were significantlylower than that in the vehicle control group.

TABLE 7 Effects of OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated on blood glucose excursionfollowing an oral glucose tolerance test in ob/ob mice Dose of OXMpeptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) at position 39Glucose AUC (mg*min/dL) is amidated (nmole/kg) MEAN SEM 0 (Vehicle)32885  5341 7.5 12103* 1864 15 14837* 2322 Data given as area under theglucose curve (= integrated values from t + 0 to 120 min) (n = 7) Thesedata show that the OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated significantly reduced the bloodglucose excursion following an oral glucose tolerance test in ob/obmice. Statistical significance evaluated by Dunnett's test. (*p < 0.05vs. vehicle)

TABLE 8 Effects of OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated on blood glucose excursionfollowing an intraperitoneal (ip) glucose tolerance test in ob/ob miceDose of OXM peptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) atposition 39 Glucose AUC (mg*min/dL) is amidated (nmole/kg) MEAN SEM 0(Vehicle) 37894  1482 7.5 16944* 1821 Data given as area under theglucose curve (= integrated values from t + 0 to 120 min) (n = 7) Thesedata show that the OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated significantly reduced the bloodglucose excursion following an intraperitoneal (ip) glucose tolerancetest in ob/ob mice. Statistical significance evaluated by Dunnett'stest. (*p < 0.05 vs. vehicle)

EXAMPLE 11 Acute Effects on Plasma Glucose, Triglyceride Levels, andHepatic Gene Expression in Male Diet-Induced Obese C57BL/6 Mice

In order to investigate metabolic pathways that are modulated bytreatment with the OXM peptide analogue of SEQ ID NO: 3 wherein theCys(PEG20k) at position 39 is amidated independent of weight lossrelated changes, the OXM peptide analogue and a positive control (a longacting GLP-1R agonist) are administered by subcutaneous injection to 3month old male diet-induced obese (DIO) mice. The day before the study,mice are randomized to treatment groups (N=7/group), each group havingsimilar mean body weight. That same night (approximately 10 PM), animalsare placed into clean cages and dosed with vehicle, 22.5 nmole/kg of theOXM peptide analogue, or 22.5 nmole/kg of the positive control bysubcutaneous injection. Food is removed at the time of injection ofpeptide or vehicle. The following morning (approximately 10 AM), theanimals are sacrificed to collect plasma and liver tissue. Plasmaglucose and triglyceride concentrations are measured using a Hitachiblood chemistry analyzer. Gene expression is determined by RT-PCR.Malonyl-CoA levels are measured by HPLC.

After a single injection, plasma glucose was significantly decreasedrelative to vehicle control in all treatment groups. Plasma triglyceridelevel was decreased relative to vehicle control only in mice treatedwith the OXM peptide analogue of SEQ ID NO: 3 wherein the Cys(PEG20k) atposition 39 is amidated but not in those treated with the long-actingGLP-1R agonist. In addition, hepatic pgc-1α gene expression wassignificantly increased by 45%, and liver malonyl-CoA concentrationswere significantly decreased by 30% in mice treated with the OXM peptideanalogue of SEQ ID NO: 3 wherein the Cys(PEG20k) at position 39 isamidated relative to vehicle controls. The OXM peptide analogue of SEQID NO: 3 wherein the Cys(PEG20k) at position 39 is amidatedsignificantly decreased hepatic PCSK9 gene expression relative tovehicle controls.

1. An Oxyntomodulin peptide analogue comprising the amino acid sequence:(SEQ ID NO: 5) His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Xaa₃₈-Xaa₃₉

wherein Xaa₃₈ is Cys, Cys-PEG, or is absent; Xaa₃₉ is Cys, Cys-PEG, oris absent; and wherein the C-terminal amino acid is optionally amidated.2. The Oxyntomodulin peptide analogue according to claim 1, comprisingthe amino acid sequence: (SEQ ID NO: 2)His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg- Asn-Asn-Ile-Ala-Cys-Cys

wherein the Cys residue at position 38 is optionally PEGylated; andwherein the Cys residue at position 39 is optionally PEGylated; and thecarboxyl group of the Cys at position 39 is optionally amidated.
 3. TheOxyntomodulin peptide analogue according to claim 2, wherein theanalogue is PEGylated on the thiol of the Cys residue at either position38 or position 39 with an approximately 40 kDa PEG molecule.
 4. TheOxyntomodulin peptide analogue according to claim 2, wherein theanalogue is PEGylated on the thiol of both Cys residues at positions 38and 39 with an approximately 20 kDa PEG molecule in each case andcomprises the amino acid sequence: (SEQ ID NO: 3)His-(D-Ser)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-(1-Nal)-Ser-Lys-Tyr-Leu-Asp-Glu-Lys-Ala-Ala-Gln-Glu-Phe-Val-Gln-Trp-Leu-Leu-Asn-(Aib)-Ala-Arg-Asn-Arg-Asn-Asn-Ile-Ala-Cys(PEG20k)-Cys(PEG20k)

wherein the carboxyl group of the Cys residue at position 39 isoptionally amidated.
 5. The Oxyntomodulin peptide analogue according toclaim 4, wherein the PEG molecule is linear.
 6. The Oxyntomodulinpeptide analogue according to claim 5, wherein the carboxyl group of theCys residue at position 39 is amidated.
 7. The Oxyntomodulin peptideanalogue according to claim 1, wherein the Cys residue at position 39 isabsent, and the Cys residue at position 38 is PEGylated with anapproximately 40 kDa PEG molecule and is optionally amidated.
 8. Apharmaceutical composition comprising the Oxyntomodulin peptide analogueof claim 1, and a pharmaceutically acceptable carrier, diluent, orexcipient.
 9. A method of treating non-insulin-dependent diabetes orobesity in a subject in need thereof, comprising administering to thesubject in need thereof an effective amount of an Oxyntomodulin peptideanalogue according to claim
 1. 10. The method according to claim 9,wherein the method of treating is non-insulin-dependent diabetes. 11.The method according to claim 9, wherein the method of treating isobesity.
 12. A pharmaceutical composition comprising the Oxyntomodulinpeptide analogue of claim 6, and a pharmaceutically acceptable carrier,diluent, or excipient.
 13. A method of treating non-insulin-dependentdiabetes or obesity in a subject in need thereof, comprisingadministering to the subject in need thereof an effective amount of anOxyntomodulin peptide analogue according to claim
 6. 14. The methodaccording to claim 13, wherein the method of treating isnon-insulin-dependent diabetes.
 15. The method according to claim 13,wherein the method of treating is obesity.